CN116376896A - A kit, preparation method and extraction method for extracting plasma free DNA - Google Patents

Abstract

The present invention relates to a kit, a preparation method and an extraction method for extracting plasma free DNA, comprising: 1. Proteinase K: The mass/volume ratio of proteinase K in plasma samples is 5:1‑10:1, and the concentration range of proteinase K is 10mg/mL‑30mg/mL; lysis buffer: 1‑4.5M guanidine isothiocyanate , 1‑6mM EDTA, 20‑150mM Tris‑HCl, 0.05%‑20% NP40, pH 4‑6; binding buffer: 1‑4M guanidine isothiocyanate, 1‑3M guanidine hydrochloride, 20‑150mM Tris‑ HCl, 1‑6mM EDTA, 0.05%‑30% Tween20, 10%‑30% PEG 8000, 10%‑35% isopropanol, pH 4‑6; Wash Buffer 1: 1‑3M Guanidine HCl, 20‑ 150mM Tris‑HCl, 0.05%‑30% Tween20, 10%‑35% ethanol, pH 5‑7; Wash Buffer 2: 20‑150mM Tris‑HCl, 1‑6mM EDTA, 30%‑60% None Water ethanol, pH 5‑8; elution buffer is one of enzyme-free water and sterilized water. The invention is simple and fast in operation, can perform micro-extraction, has high purity of extracted nucleic acid, does not contain detection inhibitors, and has low cost.

Description

A kit, preparation method and extraction method for extracting plasma free DNA

technical field

The invention relates to the field of biotechnology, in particular to a kit for extracting plasma free DNA, a preparation method and an extraction method.

Background technique

Free DNA (cell-free DNA, cfDNA) is a kind of DNA free from cells in blood or body fluids. In 1947, Mandel and Metais first reported the presence of cfDNA in peripheral blood. Under physiological conditions, cfDNA in the blood mainly comes from the necrosis and apoptosis of white blood cells. In some diseases and special conditions, other cells will also release free DNA into the plasma, including circulating tumor DNA (circulating tumor DNA) from tumor cells. , ctDNA), cfDNA derived from the fetus, etc. In 1994, Vasioukhin et al. and Sorenson et al. found mutations of the RAS gene in peripheral blood ctDNA of patients with acute myeloid lymphoma, myelodysplastic syndrome, and pancreatic cancer, indicating that ctDNA can be used to discover tumor-specific gene mutations. With the continuous emergence of new research results, the potential value of cfDNA in the early diagnosis of diseases, prognosis evaluation, and efficacy monitoring has attracted more and more attention. The detection of cfDNA in plasma or serum can be regarded as a kind of "liquid biopsy", which is conducive to various related diagnoses and can avoid invasive tissue biopsy. Using blood as a test sample makes it possible to collect test samples repeatedly, which is conducive to tracking and monitoring the development of the disease and the treatment process of the tumor.

At present, cfDNA detection requires extremely high methodology. Although the current cfDNA detection methods have high sensitivity and specificity, there are still some inconsistencies with traditional pathological or cytogenetic detection results. Since the target cfDNA in the plasma is negligible, improper collection of specimens, use of anticoagulants and storage will affect the quality of the test, resulting in false positive or false negative results. Therefore, the extraction and detection methods should be optimized to further improve the detection of cfDNA. Sensitivity and specificity, shortening the time of detection, reducing the cost of detection, and at the same time achieving standardization among detection methods are all problems that need to be solved urgently.

Contents of the invention

The object of the present invention is to provide a kit, preparation method and extraction method technology for extracting plasma free DNA, so as to solve the problems raised in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions:

In the first aspect, the present invention provides a DNA nucleic acid extraction kit from a stool sample, the kit mainly includes the following components: proteinase K, lysis buffer, binding buffer, rinsing buffer 1, rinsing buffer 2, elution buffer .

Proteinase K:

Proteinase K belongs to the S8 family in the SB superfamily of serine proteases. It consists of a peptide chain with high cleavage activity. The substrate recognition site can form a 3-strand antiparallel β sheet with the substrate, which can catalyze aliphatic Carboxy-terminal peptide bond cleavage of amino acids and aromatic amino acids. The enzyme is purified to remove RNase and DNase activity. Proteinase K is active in a wide pH range of pH 4-12.5, and the action temperature range is 37-65°C.

The mass/volume ratio of proteinase K in the plasma sample to proteinase K is 5:1-10:1, and the concentration range of proteinase K is 10mg/mL-30mg/mL.

Preferably, the volume ratio of the sample to be treated to the proteinase K is 10:1, and the concentration range of the proteinase K is 20 mg/mL.

Lysis buffer:

The lysis buffer includes: 1-4.5M guanidine isothiocyanate, 1-6mM EDTA, 20-150mM Tris-HCl, 0.05%-20% NP40, pH 4-6.

Preferably, the content of components in the lysis buffer is: 4M guanidine isothiocyanate, 6mM EDTA, 100mM Tris-HCl, 10% NP40, and the pH of the lysis solution is 5.5.

When the lysis buffer provided by the present invention lyses a plasma sample, the nucleic acid in the sample is freed in the lysis system, and the separation of nucleic acid and protein is promoted, thereby releasing the nucleic acid.

The specific principles are as follows:

Guanidine isothiocyanate: white crystalline powder, guanidine isothiocyanate is a powerful protein denaturant, without RNase and DNase activity. In nucleic acid extraction, guanidine isothiocyanate can rapidly break cells or viruses to release nucleic acids, inhibit nucleases released from cells, and ensure the integrity of the primary structure of nucleic acids.

EDTA: Ethylenediaminetetraacetic acid, which can combine with metal ions such as Mg2+, Ca2+, Mn2+, Fe2+, etc., can prevent metal ions from activating protease, thereby reducing the impact of metal ions on nucleic acid quality.

Tris-HCl: helps to keep the pH stable during cell lysis and extraction, maintain the charged state of the nucleic acid phosphate group and the silica membrane, facilitate the maintenance of hydrogen bonds, and prevent the nucleic acid from being destroyed.

NP40: It is a mild non-ionic detergent, 1% concentration can basically destroy the cell membrane, but the damage to the nuclear membrane is weak, combined with a specific buffer can obtain cytoplasmic proteins. It has a strong binding force with protein and is used to prevent the interaction between the hydrophobic molecules of the substance and ensure the full dissolution and structural stability of the protein. Especially for solubilization under non-denaturing conditions of membrane proteins to release nucleic acids.

binding buffer

Binding buffer, including: 1-4M guanidine isothiocyanate, 1-3M guanidine hydrochloride, 20-150mM Tris-HCl, 1-6mM EDTA, 0.05%-30% Tween20, 10%-30% PEG 8000, 10%- 35% isopropanol, pH 4-8.

Preferably, the content of the components in the binding buffer is: 4M guanidine isothiocyanate, 2M guanidine hydrochloride, 100m Tris-HCl, 4mMEDTA, 0.10% Tween20, 10% PEG 8000, 30% isopropanol, and its pH is 7.5.

The specific principles are as follows:

Guanidine Hydrochloride: It is a strong inhibitor of nuclease, but not a strong denaturant. Its main function in nucleic acid extraction is to denature proteins and inhibit enzyme activity. Guanidine hydrochloride can quickly destroy the cell membrane, denature and precipitate proteins, so that nucleic acids can get rid of the entanglement of proteins.

Tween20 (Tween): It is a non-ionic surfactant used to destroy cell membranes (lyse cells) to release soluble substances in cells. They can disrupt protein-protein, protein-lipid, and lipid-lipid connections, structurally denature proteins, and remove proteins that interact with nucleic acids.

PEG 8000 (Polyoxyethylene 8000): It is prone to oxidative degradation in the air, and it should be avoided to expose it to the air and or high temperature environment. After activation, it can be combined with peptides or proteins to precipitate proteins. The principle is through spatial repulsion, The protein is forced to gather together and cause precipitation.

Isopropanol: It can capture the moisture around RNA/DNA, so RNA/DNA can aggregate and precipitate.

Wash buffer 1

Wash buffer 1, including: 1-3M guanidine hydrochloride, 20-150mM Tris-HCl, 0.05%-30% Tween20, 10%-35% absolute ethanol, pH 5-8.

Preferably, the composition of the washing buffer 1 is: 2M guanidine hydrochloride, 50mM Tris-HCl, 10% Tween20, 35% ethanol, and the pH is 8.0.

Wash buffer 2

Wash buffer 2, including: 20-150mM Tris-HCl, 1-6mM EDTA, 30%-60% absolute ethanol, pH 5-8.

Preferably, the composition of the washing buffer 2 is: 100 mM Tris-HCl, 4 mM EDTA, 60% ethanol, pH 8.0. It is used to wash away residual salt ions and guanidinium salts, so as not to have adverse effects on subsequent molecular biology experiments after nucleic acid extraction.

Elution buffer

The elution buffer is one of enzyme-free water and sterilized water.

In a second aspect, the present invention provides a method for preparing a kit for extracting plasma free DNA, comprising the following steps:

A. Obtain proteinase K;

B. Preparation of lysis buffer: add the guanidine isothiocyanate, Tris-HCl, EDTA and NP40 in deionized water, and dissolve and mix the components to obtain a mixture, adjust the pH of the mixture to 4- 6;

C. Preparation of binding buffer: add the guanidine isothiocyanate, guanidine hydrochloride, Tris-HCl, EDTA, PEG 8000, isopropanol and Tween-20 in deionized water, and dissolve and mix the components to obtain mixture, adjusting the pH of the mixture to 4-8.

D. Preparation of rinsing buffer 1: add the guanidine hydrochloride, Tris-HCl, Tween20, absolute ethanol to deionized water, and dissolve and mix the components to obtain a mixture, adjust the pH of the mixture to 5- 8.

E. Preparation of rinsing buffer 2: adding the Tris-HCl, EDTA and absolute ethanol to deionized water, and dissolving and mixing each component to obtain a mixture, and adjusting the pH of the mixture to 5-8.

F. The elution buffer is one of enzyme-free water and sterilized water.

third aspect. The invention provides an extraction method of a kit for extracting plasma free DNA, comprising the following steps:

A. Sample Lysis

Plasma or sample: add 1ml serum/plasma sample to a centrifuge tube (prepared by yourself), if the sample is less than 1ml, add PBS solution to make up to 1ml volume, add 100ul proteinase K to the above solution, mix well, add 800ul lysis buffer, Shake vigorously for at least 30 seconds;

B. Adsorption binding

Incubate at 60°C for 30 minutes, mix by inverting, add 1800ul of binding buffer, invert and mix 10 times or shake vigorously for 15-30 seconds, bathe in ice for 5 minutes, connect the negative pressure device correctly, insert the connecting tube into the negative pressure device On the socket, insert the adsorption column into the connecting tube, insert the extension tube into the adsorption column with the cover removed, add all the mixed solution after the ice bath into the extension tube, turn on and adjust the negative pressure to -900~-800mbar, slowly suck Take the solution in the tube. After the solution is completely absorbed, turn off the negative pressure switch, and when the pressure returns to 0mbar, carefully remove the extension tube;

C. Rinse

Add 500ul rinse buffer 1 to the adsorption column, turn on and adjust the negative pressure to -900～-800mbar, turn off the negative pressure switch after the solution is completely absorbed; add 750ul rinse buffer 2 to the adsorption column, turn on and adjust the negative pressure To -900 ~ -800mbar, when the solution is completely sucked, turn off the negative pressure switch; add 750ul absolute ethanol to the adsorption column, turn on and adjust the negative pressure to -800 ~ -900mbar, when the solution is completely sucked, turn off the negative pressure switch ;When the pressure returns to 0mbar, remove the adsorption column, place it in a new collection tube, centrifuge at 12,000rpm for 3 minutes, and pour off the waste liquid in the collection tube;

D. Elution

Allow the column to dry completely at room temperature for several minutes. Place the adsorption column in a new 1.5ml centrifuge tube, add 20-150ul of elution buffer to the middle of the adsorption column, place at room temperature for 3 minutes, centrifuge at 12,000rpm for 1 minute, collect the DNA solution, and store the DNA at -20°C.

Compared with prior art, the beneficial effect that the present invention reaches is:

(1) The operation is simple and fast, and can be used for micro-extraction: the kit can be used with a negative pressure device, which is easy to operate, saves the traditional centrifugation step, and directly uses the negative pressure device for extraction, which can quickly and efficiently extract free nucleic acids in plasma , so that a small amount of plasma samples can be used to obtain nucleic acids with higher purity and yield, which can meet the amount of subsequent nucleic acid detection.

(2) The extracted nucleic acid has high purity and does not contain detection inhibitors: the extracted nucleic acid does not contain nucleases and detection inhibitors, and the DNA is stored for a long time. It is subsequently used in molecular biology experiments such as PCR, fluorescent quantitative PCR and next-generation sequencing, which is beneficial Preservation and detection of nucleic acid in plasma samples.

(3) Low cost: All components of the kit are self-developed and mass-produced. The cost is low, and it can meet the research and application in multiple fields of molecular biology. It has broad application prospects and market promotion value.

Description of drawings

Fig. 1 is the enrichment effect figure of adsorption column of different batches of extraction reagent of the present invention;

Fig. 2 is the extraction effect diagram of different batches of extraction reagents of the present invention;

Figure 3 is a comparison chart of cfDNA concentration extracted from the experimental group kit (Kangwei) and the comparison group (Qiajie) of the present invention;

Fig. 4 is a comparison chart of cfDNA fluorescence quantification extracted from the experimental group kit (Kangwei) and the comparison group (Qiajie) of the present invention;

Fig. 5 is the comparison diagram of the libraries of the experimental group kit (Kangwei) and the comparison group (Qiajie) of the present invention;

Fig. 6 is a comparison chart of the sequence mapping between the experimental group kit (Kangwei) and the comparison group (Qiagen) of the present invention;

Fig. 7 is the comparison chart of the sequencing rmdup of the experimental group kit (Kangwei) and the comparison group (Qiagen) of the present invention;

Detailed ways

In order to understand the characteristics and technical content of the present invention in more detail, the implementation of the present invention will be described in detail below in conjunction with the accompanying drawings. The attached drawings are only for reference and description, and are not intended to limit the present invention.

Example 1.

This embodiment provides a plasma sample DNA nucleic acid extraction kit and a preparation method. The kit includes proteinase K, lysis buffer, binding buffer, washing buffer 1, washing buffer 2, and elution buffer.

In this embodiment, each reagent component of the kit is as follows:

Proteinase K is 20mg/mL;

Lysis buffer: 1-4.5M guanidine isothiocyanate, 1-6mM EDT, 20-150mM Tris-HCl 0.05%-20% NP40, pH 5.5;

Binding buffer: 4M isothiocyanate, 2M guanidine hydrochloride, 100m Tris-HCl, 4mM EDTA, 0.10% Tween20, 10% PEG 8000, 30% isopropanol, the pH is 7.5;

Wash buffer 1: 2M guanidine hydrochloride, 50mM Tris-HCl, 10% Tween20, 35% ethanol, pH 8.0;

Wash buffer 2: 100mM Tris-HCl, 4mM EDTA, 60% ethanol, pH 8.0;

The elution buffer is one of enzyme-free water and sterilized water.

Example 2. Plasma sample DNA extraction method (column method extraction)

The plasma extraction kit reagents prepared in Example 1 were used to extract DNA and nucleic acid from plasma samples.

The specific steps of column method extraction are as follows:

1. Sample Lysis

Plasma or sample: add 1ml serum/plasma sample to a centrifuge tube (prepared by yourself), if the sample is less than 1ml, add PBS solution to make up to 1ml volume. Add 100ul proteinase K to the above solution and mix well. Add 800ul lysis buffer and shake vigorously for at least 30 seconds.

2. Adsorption binding

Incubate at 60°C for 30 minutes, mixing by inverting several times. Add 1800ul binding buffer, invert and mix 10 times or shake vigorously

15-30 seconds. Ice bath for 5 minutes. Correctly connect the negative pressure device, and insert the connecting tube into the socket of the negative pressure device. Insert the adsorption column into the connecting tube. Insert the extension tube into the decapped adsorption column. Add all the mixed solution after ice bath into the extension tube, turn on and adjust the negative pressure to -900~-800mbar, and slowly suck the solution in the tube. After the solution is completely absorbed, turn off the negative pressure switch, and when the pressure returns to 0mbar, carefully remove the extension tube.

3. Rinse

Add 500ul of washing buffer 1 to the adsorption column, turn on and adjust the negative pressure to -900~-800mbar, and turn off the negative pressure switch after the solution is completely sucked away. Add 750ul of washing buffer 2 to the adsorption column, turn on and adjust the negative pressure to -900~-800mbar, and turn off the negative pressure switch after the solution is completely sucked away. Add 750ul of absolute ethanol to the adsorption column, turn on and adjust the negative pressure to -800~-900mbar, and turn off the negative pressure switch after the solution is completely sucked away. When the pressure returned to 0 mbar, the adsorption column was removed, placed in a new collection tube, centrifuged at 12,000 rpm for 3 minutes, and the waste liquid in the collection tube was discarded.

4. Elution

Allow the column to dry completely at room temperature for several minutes. Place the adsorption column in a new 1.5ml centrifuge tube, add 20-150ul of elution buffer to the middle of the adsorption column, place at room temperature for 3 minutes, centrifuge at 12,000rpm for 1 minute, collect the DNA solution, and store the DNA at -20°C.

Example 3. Test the extraction effect of different adsorption columns

Experimental materials: plasma samples; plasma sample extraction reagents prepared in Example 1, wherein the adsorption columns were selected from domestic adsorption column 1, domestic adsorption column 2 and imported adsorption column

Experimental method: Take 4 equal parts (1ml each) of fresh plasma samples and divide them into clean centrifuge tubes, add 100μl proteinase K, mix well, add 800μl lysis buffer, shake vigorously for at least 30 seconds. Incubate at 60°C for 30 minutes, mixing by inverting several times. Add 1800μl binding buffer, mix by inverting 10 times or shake vigorously for 15-30 seconds. After 5 minutes of ice bathing, connect the negative pressure device, use three kinds of adsorption columns for nucleic acid adsorption, rinse buffer 1, rinse buffer 2, and absolute ethanol respectively, then centrifuge and dry, and add elution buffer to elute.

The 4 samples were divided into three groups, which were subjected to three extraction treatments: (domestic adsorption column group 1) 4 of them were extracted with Jiangsu Kangwei Century's domestic adsorption column; (domestic adsorption column group 2) 4 plasma samples were extracted with American Bio-based domestic adsorption column for extraction; (imported adsorption column group) 4 plasma samples were extracted with thermo adsorption column.

The extracted DNA was detected by Qubit2.0 to detect the concentration of nucleic acid samples. The results are shown in Table 1. The extraction effect of the domestic adsorption column group is basically the same, and the concentration of nucleic acid extracted by the imported adsorption column extraction group is slightly higher.

Table 1. Comparison of extraction effects of different adsorption columns

Embodiment 4. Test the extraction effect of different batches of reagents

Experimental materials: plasma samples; plasma sample extraction reagents (different batches) prepared in Example 1, wherein the adsorption column is selected from domestic adsorption column 1

Experimental method: Samples were collected from standard DNA Marker mixed serum, and two production batches of domestic adsorption columns 1 and extraction reagents were used for DNA enrichment and separation. The Qsep100TM automatic nucleic acid and protein analysis system was used to compare the two batches. The enrichment and separation results were highly consistent among different batches. As shown in Figure 1 and Figure 2.

Example 5. Plasma Sample Extraction Kit Extraction Ability Test

Experimental materials: plasma samples provided by 3 volunteers.

Experimental group reagent: plasma sample extraction reagent prepared in Example 1 (adopting domestic Kangwei adsorption column)

Comparative reagents: QIAamp Circulating Nucleic Acid Kit (50)./ID:55114

Experimental method: The plasma samples provided by 3 volunteers were stored in plasma cell-free DNA preservation tubes (Kangwei Century, Cat. No. CWY055)

(A) Experimental group reagent-negative pressure device extraction group: take two fresh plasma samples and put them in clean centrifuge tubes, and extract 5ml/8ml respectively,

5ml extraction: add 1ml proteinase K, mix well, add 4ml lysis buffer, shake vigorously for at least 30 seconds. Incubate at 60°C for 30 minutes, mixing by inverting several times. Add 9ml binding buffer, mix by inverting 10 times or shake vigorously for 15-30 seconds. After 5 minutes of ice bathing, connect the negative pressure device, use three kinds of adsorption columns for nucleic acid adsorption, wash with rinsing buffer 1, rinsing buffer 2, and absolute ethanol respectively, then centrifuge and dry, and add elution buffer for elution.

8ml extraction: add 1.6ml proteinase K, mix well, add 6.4ml lysis buffer, shake vigorously for at least 30 seconds. Incubate at 60°C for 30 minutes, mixing by inverting several times. Add 14.4ml binding buffer, invert and mix 10 times or vigorously shake for 15-30 seconds. After 5 minutes of ice bathing, connect the negative pressure device, use three kinds of adsorption columns for nucleic acid adsorption, wash with rinsing buffer 1, rinsing buffer 2, and absolute ethanol respectively, then centrifuge and dry, and add elution buffer for elution.

(B) Reagents of the comparison group - negative pressure device extraction group: take two fresh plasma samples and divide them into clean centrifuge tubes, and extract 5ml/8ml respectively, using Qiagen's free DNA extraction kit (catalogue number 55114) Extract according to the instructions.

The extracted nucleic acid was tested for DNA concentration with Qubit2.0, and the experimental results are shown in Table 2. Compared with the comparison group-negative pressure device extraction group, the extraction concentration of the experimental group-negative pressure device extraction group was slightly better, as shown in Figure 3 Show.

Subsequent fluorescent quantitative PCR detection of KTB primers, compared with the experimental group-negative pressure device extraction group and the control group-negative pressure device extraction group, the experimental results are shown in Table 3, and the CT value of the experimental group is slightly lower, as shown in Figure 4.

After the extraction was completed, the CW3045S/CW3026S Kangwei Library Extraction Kit was used for library construction, and the input amount was 20ul, and the Huada MGI platform was sequenced. Table 4 shows the concentration of the library measured by Qubit2.0 after library construction. As shown in Figure 5, the experimental group The extraction concentration of the extraction reagent was slightly better than that of the comparison group, and the mapped and rmdup values of the sequencing results were also slightly better, as shown in Figure 6 and Figure 7.

However, the column method used in the experimental group plasma nucleic acid extraction reagents and methods provided in this example has a relatively high extraction concentration, a slightly lower Ct value in PCR detection, and a slightly higher concentration out of the warehouse, which can better meet the requirements of subsequent nucleic acid detection experiments. .

Table 2. Extraction concentration of plasma extraction kit

Table 3. Fluorescence quantitative comparison of plasma extraction kits

Table 4. Comparison of libraries extracted by plasma extraction kits

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (3)

1. A kit for extracting plasma free DNA, comprising: proteinase K: the mass/volume ratio of proteinase K in the plasma sample was 5:1-10:1, the concentration range of proteinase K is 10mg/mL-30mg/mL; lysis buffer: 1-4.5M guanidine isothiocyanate, 1-6mM EDTA, 20-150mM Tris-HCl, 0.05% -20% NP40, pH 4-6; binding buffer: 1-4M guanidine isothiocyanate, 1-3M guanidine hydrochloride, 20-150mM Tris-HCl, 1-6mM EDTA, 0.05-30% Tween20, 10-30% PEG 8000, 10-35% isopropanol, and pH 4-8; rinse buffer 1:1-3M guanidine hydrochloride, 20-150mM Tris-HCl, 0.05-30% Tween20, 10-35% absolute ethyl alcohol, and the pH value is 5-8; rinse buffer 2:20-150mM Tris-HCl, 1-6mM EDTA, 30-60% absolute ethyl alcohol, pH 5-8; the eluting buffer is one of enzyme-free water and sterilized water.

2. The preparation method of the kit for extracting plasma free DNA is characterized by comprising the following steps:

A. obtaining proteinase K;

B. preparation of lysis buffer: adding the guanidine isothiocyanate, tris-HCl, EDTA and NP40 into deionized water, dissolving and uniformly mixing the components to obtain a mixture, and regulating the pH of the mixture to 4-6;

C. preparation of binding buffer: adding the guanidine isothiocyanate, guanidine hydrochloride, tris-HCl, EDTA, PEG 8000, isopropanol and Tween-20 into deionized water, dissolving and uniformly mixing the components to obtain a mixture, and regulating the pH of the mixture to 4-8;

D. rinse buffer 1 preparation: adding guanidine hydrochloride, tris-HCl, tween20 and absolute ethyl alcohol into deionized water, dissolving and uniformly mixing the components to obtain a mixture, and regulating the pH value of the mixture to 5-8;

E. rinse buffer 2 preparation: adding Tris-HCl, EDTA and absolute ethyl alcohol into deionized water, dissolving and uniformly mixing the components to obtain a mixture, and regulating the pH value of the mixture to 5-8;

F. the eluting buffer is one of enzyme-free water and sterilized water.

3. An extraction method of a kit for extracting plasma free DNA, which is characterized by comprising the following steps:

A. sample lysis

Plasma or sample: adding 1ml serum/plasma sample into a centrifuge tube (self-contained), if the sample is less than 1ml, adding PBS solution to compensate to 1ml volume, adding 100ul proteinase K into the above solution, uniformly mixing, adding 800ul lysis buffer, and shaking vigorously for at least 30 seconds;

B. adsorption bonding

Incubating at 60 ℃ for 30 minutes, mixing the materials upside down, adding 1800ul of binding buffer solution, mixing the materials upside down for 10 times or shaking the materials vigorously for 15-30 seconds, ice-bathing for 5 minutes, correctly connecting a negative pressure device, inserting a connecting pipe into a socket of the negative pressure device, inserting an adsorption column into the connecting pipe, inserting an extension pipe into the adsorption column with a cover opened, adding all the mixed solution after ice-bathing into the extension pipe, starting and adjusting the negative pressure to-900 to-800 mbar, and slowly sucking the solution in the pipe. When the solution is completely sucked away, the negative pressure switch is closed, and when the pressure is restored to 0mbar, the extension tube is carefully removed;

C. rinsing

Adding 500ul of rinsing buffer solution 1 into the adsorption column, starting and adjusting the negative pressure to-900 to-800 mbar, and closing a negative pressure switch when the solution is completely absorbed; adding 750ul rinsing buffer solution 2 into the adsorption column, starting and adjusting the negative pressure to-900 to-800 mbar, and closing a negative pressure switch when the solution is completely absorbed; adding 750ul absolute ethyl alcohol into the adsorption column, starting and adjusting the negative pressure to-800 to-900 mbar, and closing the negative pressure switch when the solution is completely absorbed; when the pressure was restored to 0mbar, the adsorption column was removed and placed in a fresh collection tube, which was centrifuged at 12,000rpm for 3 minutes, and the waste liquid in the collection tube was discarded;

D. elution

The column was left at room temperature for several minutes to dry thoroughly. Placing the adsorption column in a new 1.5ml Centrifuge Tube (Centrifuge Tube), suspending the middle part of the adsorption column, adding 20-150ul elution buffer, standing at room temperature for 3 min, centrifuging at 12,000rpm for 1 min, collecting DNA solution, and preserving DNA at-20deg.C.

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